Exogenous abscisic acid treatment regulates protein secretion in sorghum cell suspension cultures

ABSTRACT Drought stress adversely affects plant growth, often leading to total crop failure. Upon sensing soil water deficits, plants switch on biosynthesis of abscisic acid (ABA), a stress hormone for drought adaptation. Here, we used exogenous ABA application to dark-grown sorghum cell suspension cultures as an experimental system to understand how a drought-tolerant crop responds to ABA. We evaluated intracellular and secreted proteins using isobaric tags for relative and absolute quantification. While the abundance of only ~ 7% (46 proteins) intracellular proteins changed in response to ABA, ~32% (82 proteins) of secreted proteins identified in this study were ABA responsive. This shows that the extracellular matrix is disproportionately targeted and suggests it plays a vital role in sorghum adaptation to drought. Extracellular proteins responsive to ABA were predominantly defense/detoxification and cell wall-modifying enzymes. We confirmed that sorghum plants exposed to drought stress activate genes encoding the same proteins identified in the in vitro cell culture system with ABA. Our results suggest that ABA activates defense and cell wall remodeling systems during stress response. This could underpin the success of sorghum adaptation to drought stress.

Dehydration-induced gene expression leads to a change in the proteome and metabolome, with notable enrichment of molecules with protective functions against the primary and secondary effects of drought stress. 13For example, enzymatic and non-enzymatic antioxidants, osmoprotectants, late embryogenesis abundant proteins, and molecular chaperones accumulate as an effective countermeasure to osmotic stress. 13,14,16 key question emerging from this research is why most plant species remain sensitive to drought stress when they can activate these molecular responses.Much of this research has been conducted using drought-sensitive Arabidopsis. 17Analysis of drought-tolerant species, such as sorghum, 18,19 could lead to new insights into the molecular signals that underpin field success against drought stress.
][42][43] Nevertheless, several plant proteomic studies have investigated secretome responses to osmotic, heat and high salinity stresses in diverse plant species, 42,44,45 and the critical roles of these ECM proteins in stress response are unquestionable.Many of these studies have also utilized plant cell suspension cultures due to the inherent advantages of such model systems in secreted protein analyses. 42,44Therefore, this study investigated the differential expression profiles of total soluble and secreted proteins in sorghum cell suspension cultures following ABA treatment.

ABA treatments of sorghum cell suspension cultures
White sorghum (Sorghum bicolor) cell suspension cultures 46 were used in this study.The cell suspension cultures were subcultured and maintained as described previously. 46,47All treatments were carried out on 8-day-old cell cultures, which corresponded to the mid-log phase. 47On day 8 post subculture, four biological replicate flasks were split into two 30 mL subcultures each for the control and ABA treatment to account for technical variation between samples.For ABA treatment, the cell suspension cultures were treated with a final concentration of 100 μM ABA using a filter-sterilized 0.1 M ABA (Catalog No. A1049, Sigma Aldrich, Saint Louis, USA) stock solution prepared in 70% methanol.The control cells were spiked with an equivalent volume of filter-sterilized 70% methanol.Cells of both treatment groups were incubated for 72 h during which cell viability 48,49 and fresh/dry weight growth measurements were taken.The 0-h time point denotes the time immediately after treatment was imposed.For both cell viability and growth measurements, four biological replicate cell cultures were used for each treatment.

Total soluble and secreted protein extraction
Four biological replicate control and ABA-treated cell cultures were harvested at 72 h post treatment for total soluble and secreted protein extraction as previously described. 50Briefly, cell suspension cultures were filtered through four layers of Miracloth to separate the cells from the medium.The cells were subsequently washed with sterile distilled water and stored at −80°C, while the medium was centrifuged at 2 500 � g for 10 minutes to collect the cell-free supernatant.Total soluble protein (TSP) was extracted from homogenized cells, while secreted proteins were extracted from the cell-free culture medium by acetone precipitation and centrifugation.The resultant protein pellets were solubilized in appropriate volumes of extraction buffer (7 M urea, 2 M thiourea, 4% (w/ v) 3-(3-cholamidopropyl)dimethylammonio)-1-propanesulfonate) with vigorous vortexing overnight. 50The extracted TSP and secreted proteins were quantified and prepared for isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass spectrometry analysis.[52]

iTRAQ labelling, LC-MS/MS, protein identification, and quantification
Aliquots of 12.5 μg protein from each sample were processed for labeling using an iTRAQ Reagent-Multiplex Buffer Kit (AB Sciex, Redwood City, CA, USA) following the manufacturer's instructions.The protein samples were then digested with trypsin overnight 51 and peptides subsequently labeled using an 8-plex iTRAQ reagent kit (AB Sciex) according to the manufacturer's instructions.The 4-replicate control samples of TSP and secreted protein fractions were separately labeled with iTRAQ tags 113, 114, 115 and 116, while ABA-treated samples were labeled with tags 117, 118, 119 and 121.This gave rise to two separate iTRAQ experiments each with the control and ABA-treated samples of each proteome.After labeling, all TSP samples were pooled (across the 8 tags) as was also done for the secreted samples.The pooled iTRAQ-labeled samples were subsequently cleaned-up using hydrophobic interaction chromatography (HILIC) solid phase extraction (SPE) cartridges (PolyLC Inc., Columbia, MD, USA) as described previously. 52Then LC-MS/MS and mass spectrometric analyses were conducted on peptides originating from 5 μg of sample following detailed protocols described in Goche et al. 52 LC-MS/MS was conducted using a Triple TOF 6600 mass spectrometer (AB Sciex) linked to an Eksigent 425 LC system via a Sciex Nanospray III source (AB Sciex).The acquisition of mass spectrometer data was done using the AB Sciex Analyst TF 1.7.1 instrument control and data processing software.
Protein identification and relative quantification were conducted using the detailed protocol described in Goche et al. 52 with minor modifications.The raw.wiff data files were processed against the UniProt protein sequences of Sorghum bicolor only (downloaded in May 2018) using the AB Sciex ProteinPilot 5.01 version 4895 software with the Paragon Algorithm 5.0.1.0.4874.The raw protein identification data were exported from ProteinPilot to Microsoft® Excel version 16.16.27for manual data-handling and filtering.All duplicate proteins and those identified based on a single peptide were manually removed from the dataset, giving rise to 707 and 257 positively identified proteins in the TSP and secreted protein fractions, respectively.For each iTRAQ experiment, the relative quantification of the ABA-responsive proteins was generated as a ratio of each protein relative to the 113-tagged control sample.Then an average ratio of each protein was computed across all four biological replicate samples.Thereafter, a Student's t-test at p ≤.05 was used to calculate the statistical significance in the ratios.

Bioinformatics analysis
Gene ontology analysis and protein family names were retrieved from the UniProt 53 and Interpro 54 databases, respectively.The signal peptide predictions were conducted on the SignalP 6.0 server. 55

Plant cell suspension cultures are a useful resource for studying ABA-response
ABA is a multifunctional phytohormone, [1][2][3][4] and its role in plant stress response has been extensively studied and reviewed. 3,14,16,56,5716 To our knowledge, however, similar investigations on the effects of exogenous ABA on secretory proteins are minimal, 37,39 yet the secretome is essential during plant cell growth, development, and stress response.[40][41][42][43] The secretome consists of proteins that are secreted into the ECM of plant cells. 42 Some revews 42,44 have summarized secretome studies of various plant species in response to biotic and abiotic stresses using whole plants or cell suspension cultures.In the current study, we used sorghum cell suspension cultures in line with other secretome studies that we have conducted. 48,51The utility of cell suspension cultures in ABA responses of total soluble proteins 24,27 and the secretome 37 has also been tested and validated in Arabidopsis (Arabidopsis thaliana), 24 rice (Oryza sativa) 27 and wheat (Triticum aestivum).37 Therefore, our white sorghum cell suspension cultures provided an experimental resource for comparing the impact of exogenous ABA on expression profiles of intracellular versus extracellular proteins.
Analysis of cellular metabolic activity (Figure S1) and fresh and dry weight (Figure S2) of sorghum cell cultures revealed no adverse effects of ABA treatment over the 72-h of exposure.The increase in cell weight from 0-72 h (Figure S2) reflected cell culture growth.Thus, the level of ABA applied, and timing of cell harvest did not compromise either cell vitality or growth, thus enabling the identification of intracellular and extracellular proteins recruited in ABA-dependent responses.

iTRAQ reveals the selective nature of protein secretion in ABA-treated sorghum cell suspension cultures
The TSP fraction was extracted from the cells, while the secreted proteins were extracted from the growth medium prior to digestion with trypsin, iTRAQ labeling and LC-MS /MS analysis.A total of 707 TSP and 257 secreted proteins were positively identified based on at least two matching peptides, and the peptide information is listed in Tables S1 and S2.The observed differences in the proportions of positively identified proteins under untreated conditions possibly reflect differences in protein diversity and function of the two cellular compartments, and the specialized and selective nature of protein secretion.][42][43][44] The iTRAQ data were subsequently analyzed using a Student's t-test at a 5% significance level to identify the ABAresponsive proteins.In total, 46 (~7%) TSP and 82 (~32%) secreted proteins were ABA-responsive (p ≤.05), as summarized in Table 1, while their iTRAQ quantitation data are given in Tables S3 and S4.A large proportion of the combined subset of ABA-responsive proteins amounting to 68%, were uncharacterized (Tables S5 and S6), as observed in other sorghum iTRAQ datasets 48,51,52 .This calls for increased experimental validation studies [59][60][61] of sorghum genes and proteins for improved functional annotations.

The extent of ABA effects on metabolism covers nearly all intracellular and extracellular compartments
We then collected bioinformatics data on signal peptide predictions, Gene Ontology (GO) terms, and protein family names to assist in assigning putative cellular locations and biological functions to these proteins (Tables S5 and S6).However, due to the extensive list of ABA-responsive proteins obtained in this study using a Student's t-test at p ≤.05 (Tables S5 and S6), we have shortened this list for illustrative purposes in Tables 2 and 3 by only showing ABA-responsive proteins based on a more stringent probability value (p ≤.01).Nevertheless, all results presentations and discussions in this study are based on the entire ABA-responsive protein selection at p ≤.05 (Tables S5 and S6).
Newly synthesized secretory proteins may be targeted for secretion via the conventional endoplasmic reticulum (ER)/ Golgi-mediated pathway. 62,63Here, N-terminal signal peptides direct proteins to the ER and Golgi apparatus for posttranslational modifications before secretion via Golgi vesicles. 62,635][66] Accordingly, signal peptide prediction results using the primary sequences of the ABA-responsive secretome (Table S6) on SignalP 6.0 55 revealed that most of the ABA-responsive secreted proteins of sorghum had predictable signal peptides (91%), while the rest were leaderless (Figure 1a; Table S6).These results are comparable to a previous sorghum secretome, where 84% of the heat-responsive secreted proteins possessed signal peptides, 48 but much higher than the 54% observed in an osmotic-stress study. 51Nonetheless, the results indicate that exogenous ABA triggers protein secretion in sorghum cell suspension cultures, as previously reported in wheat. 37xamples of N-terminal signal peptide-containing secreted proteins identified in this study include peroxidases, phytocyanins, GDSL lipase/esterases, thaumatins, germins, pathogenesis-related proteins, various proteases and their inhibitors, expansins, pectinesterases, lipid transfer proteins, leucine-rich repeat-containing proteins and several members of the glycoside hydrolase superfamily (Table S6).As reviewed by Alexandersson et al. 44 many of these proteins are members of secreted protein families under diverse environmental stresses in different plant species.Examples of leaderless ABAresponsive sorghum secreted proteins identified include members of the glycoside hydrolase family 31, UDP-glucuronosyl /UDP-glycosyltransferase, plant peroxidase, superoxide dismutase (SOD) and proteinase inhibitor II3, potato inhibitor I families (Table S6).The leaderless plant peroxidases with accessions A0A1W0W7I8 and A0A1B6QJR7 were associated with the plant cell wall and extracellular region, respectively (Table S6), thus pointing to a secretory location.On the other hand, extracellular plant SODs are known to lack signal peptides and have been reported in various plant secretome studies under pathogenic attack 64 osmotic 51 and heat 48 stresses.These signal peptide-lacking proteins of sorghum now form part of the growing list of leaderless secretory proteins in plants 62,65 that await cellular localization studies.
The signal peptide predictions of the ABA-responsive sorghum secretome (Figure 1a) were supported in part by the cellular component GO terms obtained (Figure 1b).For instance, the terms extracellular region and extracellular space were highly enriched in the secreted proteins versus the cytoplasm and cytosol locations in the ABA-responsive TSP (Figure 1b).In addition, the nucleus, proteasome complex, endoplasmic reticulum, mitochondrion, and peroxisome were exclusively identified in the ABA-responsive TSP fraction (Figure 1b).The diversity of subcellular localizations observed in the protein lists (Figure 1b; Tables S5 and S6) indicate that the extent of ABA effects on metabolism covers nearly all cell compartments.This confirms the role of ABA as a switch from normal growth to stress metabolism.

Exogenous ABA application modulates specific stress-related processes in the ECM
To understand the effects of ABA on ECM proteins, we used GO data of biological processes (Figure 2) and protein family names (Tables S6) to assign putative functions to the ABAresponsive secreted proteins (Figure 3).We then compared the functional groupings of the 46 ABA-responsive intracellular and 82 ECM proteins (Table 1) to assess whether ABA exerts a differential impact on ECM versus intracellular proteins (Figures 2 and 3; Tables S5 and S6).The results revealed that ABA modulates a variety of cellular processes in both proteomes (Figures 2 and 3).However, ABA effects on the secretome were primarily targeted toward defence/detoxification  g Signal peptide (SP) prediction results for each protein as determined by the SignalP 6.0 server (https://services.healthtech.dtu.dk/services/SignalP-6.0/).+ indicated p resence of a signal peptide, while -indicates absence of a signal peptide. h-j Gene Ontology terms for each protein as collated from the UniProt database.
k Family name as predicted using the InterPro (http://www.ebi.ac.uk/interpro/).In cases where protein families are not predicted, functional domains are listed instead.
PLANT SIGNALING & BEHAVIOR  g Signal peptide (SP) prediction results for each protein as determined by the SignalP 6.0 server (https://services.healthtech.dtu.dk/services/SignalP-6.0/).+ indicated presence of a signal peptide, while -indicates absence of a signal peptide.
h -j Gene Ontology terms for each protein as collated from the UniProt database.
k Family name as predicted using the InterPro (http://www.ebi.ac.uk/interpro/).In cases where protein families are not predicted, functional domains are listed instead.

Plant defence and detoxification
8][69][70] In this study, the ABA-responsive secretome was dominated by defense/detoxification-related proteins (32%) (Figure 3), and most were up-regulated (Table S6).Examples include plant peroxidases, thaumatins, germins, SODs and pathogenesis-related (PR) proteins with RNA nuclease (e.g.PRP-4) and chitinase (e.g.glycoside hydrolase family 18 and 19) activities (Table S6).0][71][72] For instance, plant cells under pathogen attack generate an oxidative burst at the site of infection, which is mediated by germins, SODs and peroxidases. 70,73poplastic peroxidases also generate hydrogen peroxide, which functions as a ROS signal for stress-induced gene expression 72,74 and in cell wall stiffening to prevent pathogen invasion. 70,73Other plant secretome studies have also identified various ROS antioxidant enzymes in response to heat, 48 osmotic, 51 dehydration, 75 and salt 76 stresses.About 20% of the identified ABA-responsive TSP were related to ROS detoxification, and most, including a glutathione reductase, peroxidase, thaumatin, germin, and peroxiredoxin-5-like protein, were up-regulated (Table S5) further highlighting the importance of ROS scavenging systems inside and outside the cell. 69,70,72Our results collectively support the role of ABA in enhancing plant signaling, defense and/or ROS scavenging capacities during stress response.
We also identified nine glycoside hydrolase family 18 and 19 proteins in the secretome, and all were upregulated, with fold-changes as high as 4.8 (Table S6).
Plant glucoside hydrolases metabolize various carbohydrates involved in plant cell growth, signaling and response to biotic and abiotic stresses. 77,78Members of glycoside hydrolase family 18 and 19 possess chitinase activity, and several from Arabidopsis and rice are secreted proteins. 789][80] For example, extracellular chitinases hydrolyze chitin of fungal origin, thus retarding hyphal growth and progression of fungus colonization in plants. 78,79These apoplastic chitinases may also participate in cell signaling as their chitin degradation products signal plant cells to mount further defense responses against the invading pathogen. 79Due to their induction following pathogen attack, chitinases are classified as pathogenesisrelated proteins. 71Other plant secretome studies have identified increased accumulation of chitinases in response to heat, 48,81 osmotic, 51 salt 76 and dehydration 82 stresses.Our results also add to the growing literature on the role of ABA in inducing plant chitinases, possibly as a defense mechanism against environmental stresses.

Cell wall modifying enzymes
Plant cell walls are dynamic structures that dominate the ECM and are composed of cellulose microfibrils, hemicellulose, pectin, and proteins. 58,83,84][89] The results of this study revealed that cell wall modifying enzymes (27%) were the second largest ABA-responsive functional group in the ECM as opposed to 15% in the TSP (Figure 3; Tables S5 and S6).Examples of the identified cell wall modifying enzymes in the ECM include expansins, pectinesterases, fasciclin-like arabinogalactan proteins, xyloglucan endotransglucosylase/hydrolase, and several members of glycoside hydrolases (Tables and S6).About two-thirds of these cell wall modification-related secreted proteins, including glycoside hydrolases, and XETs, were up-regulated (Table S6).2][93] Likewise, xyloglucan modification by XETs enhances cell extensibility, 87 and ABA is believed to regulate XET activity. 94Under water-limiting conditions, expansin and XET-mediated cell wall loosening enhance root growth for increased water-foraging capacity. 83,87Other secreted proteins identified include glycoside hydrolases that metabolize diverse polysaccharide components of the cell walls, such as pectin, xylan, arabinan, galactomannan, cellulose, glucan, and glycosaminoglycan (Table S6), further implicating ABA in plant cell wall biology.Numerous cell wall modifying enzymes have also been identified in other secretome studies in response to osmotic, 51 and heat, 48,81 dehydration 75,82 stresses, thus emphasizing the pivotal roles of cell walls and their remodeling during stress response.A review by Albene et al. 90 summarizes Arabidopsis cell wall proteomics studies, the major classes of cell wall protein families, and their interacting partners in cell walls.

Proteolysis
Proteolysis-related proteins were highly represented in the ABA-responsive secretome with 12 proteins (15%) but only two in the TSP (4%) (Figure 3; Tables S5 and S6).Both TSP proteins were proteasome subunits of the ubiquitinproteasome system that degrades proteins in the cytosol. 95In contrast, the secretome exhibited greater diversity of aspartic, cysteine and serine-type endopeptidases and their inhibitors, such as cystatins, a Bowman-Birk type wound-induced proteinase inhibitor WIP1, and a proteinase inhibitor II3, potato inhibitor I (Table S6).All except the proteinase inhibitor II3, potato inhibitor I possessed signal peptides, indicating that secretory proteases and protease inhibitors respond to ABA.In addition, nine of these 12 proteins were up-regulated (Table S6).Our recent review 96 and references therein project proteases and their inhibitors as critical role players in numerous physiological processes under normal growth and during stress response.Furthermore, transgenic studies using gain or loss-of -function mutants implicate proteases and/or protease inhibitors in ABA signaling processes. 96Apoplastic proteases are also regarded as defense systems against plant pathogen infection. 97,98llular transport Cellular transport is essential for the trafficking of proteins, lipids, and other molecules within and between cells for various functions.Only one protein transporting GTPase was identified in the intracellular fraction and down-regulated (Table S5).In contrast, three lipid-transporting plant nonspecific lipid-transfer protein/Par allergens were identified in the secretome, and two were up-regulated, with fold-changes of 2.41 and 4.83 (Table S6).Plant nonspecific lipid-transfer protein/ Par allergens are small, basic proteins with a wide range of functions, including lipid binding and transport.99 Their physiological roles are also diverse during plant growth and development and in response to biotic and abiotic stresses, including the thickening of the cuticle.99 Dani et al. 76 also identified two salt-inducible lipid transfer proteins in the tobacco leaf apoplastic proteome following salt stress, suggesting their role is salt-response.Our results also suggest the implication of ABA in lipid transport processes in the sorghum ECM.

Exogenous ABA regulates a broad spectrum of intracellular proteins involved in metabolism
The ABA-responsive TSP consisted of a broader selection of proteins involved in the metabolism of carbohydrates, amino acids, lipids and fatty acids, nitrogenous compounds, pigments, and lignin (Table S5).In addition, the metabolism functional group dominated the ABA-responsive TSP (43%) (Figure 3).Enzymes involved in the carbon fixation (phosphoenolpyruvate carboxylase) and glycolysis (pyruvate kinase, fructose-bisphosphate aldolase) were down-regulated (Table S5), possibly to save energy for other crucial stress-adaptive processes.Conversely, some enzymes involved in the metabolism of lipids and fatty acids (patatin, thiolase, and betahydroxyacyl-(acyl-carrier-protein) dehydratase FabZ), and lignin (cinnamyl alcohol dehydrogenase-like) were up-regulated (Table S5).In contrast, the metabolism-related functional group constituted 11% of the ABA-responsive secretome, and most were down-regulated (Figure 3).However, no theme could be drawn from the affected secreted proteins.Nevertheless, our results support the pivotal role of ABA in modulating stress metabolism of primary and secondary metabolites within and outside the cell.

Signal transduction, protein synthesis, and DNA replication are unique to the intracellular proteome
The signal transduction, protein synthesis, and DNA replication categories were exclusively present in the ABAresponsive TSP (Figure 3; Tables S5 and S6).Both Bet v I type allergens involved in the ABA-activated signaling pathway were up-regulated (Table S5).Stress sensing and signaling are critical processes that precede other plant responses to environmental changes.The Bet v I protein is a birch pollen allergen, 100 encoded by a gene family like the PYR/ PRL-1/RCARs. 3,101,102As discussed earlier, PYR/PRL-1/ RCARs are ABA receptors which bind ABA, freeing the SnRK2s from the inhibitory effects of PP2Cs.Subsequently, ABA-regulated gene expression changes are deployed, resulting in changes in cellular metabolism.Furthermore, GO annotations on the UniProt database suggest that both Bet v I type allergen proteins (accessions C5WMM0 and Q4VQB4) have molecular functions related to signal receptor activity, ABA binding, and protein phosphatase inhibitor activity (Table S5), which are similar to the functions of PYR/PRL-1/RCARs. 3The up-regulation of both Bet v I type allergen proteins in the current study (Table S5) further reinforces the positive interaction between ABA and its receptors in inactivating PP2Cs during ABA-dependent stress response. 101he up-regulation of the proliferating cell nuclear antigen (PCNA) in the intracellular (Table S5) points to the role of ABA in DNA replication.A review by Strzalka and Ziemienowicz 103 extensively details various functions of PCNA in DNA replication, DNA repair and cell cycle control, which are vital for proper cell functioning.While molecular responses to ABA and various stresses involve global changes in protein synthesis, we identified threonine-tRNA ligase class IIa as the only protein synthesis-related protein, and it was down-regulated (Table S5).It is plausible that the duration of ABA treatment may influence the types of proteins identified and their expression levels.However, time-course proteomics experiments are required to investigate the changes in the types of proteins and their accumulation patterns as a function of ABA treatment duration.
Finally, the functions of 13 ABA-responsive proteins were unclear and thus grouped as unclassified proteins (Tables S5 and S6; Figure 3).Of these proteins, 10 were identified in the secretome fraction (Table S6), possibly indicating the pool of unknown sorghum ECM proteins that may have essential roles in stress adaptation.For example, leucine-rich repeat-containing N-terminal planttype domain-containing proteins have been repeatedly identified as up-regulated proteins in response to drought in the intracellular proteome of sorghum roots, 52 and osmotic 51 and heat 48 stresses in the sorghum secretome.Therefore, such unclassified proteins may represent gene products with universal roles in abiotic stress responses that await functional validation.

The complex nature of ABA responses in the ECM versus the intracellular space
We also identified eight ABA-responsive proteins common in intracellular and secreted protein fractions (Table S7).Since all plant proteins are synthesized intracellularly before translocation to various destinations within or external to the cell, the common proteins could have been extracted before their total secretion.Alternatively, the proteins may have dual subcellular locations and/or multiple functions in plants.Furthermore, the expression levels of four of these proteins, two glycoside hydrolases (C5XKE9 and C5WXC7), a phytocyanin-like protein (C5YK12), and thaumatin (C5XN52) were statistically different between the two fractions (Table S7).As such, cellular localization and functional studies would possibly validate their subcellular location(s) and function(s) in each cell compartment and thus unravel the significance of their differential expression levels between the intracellular and extracellular fractions.

Exogenous ABA down-regulates a myriad of ECM and intracellular proteins
When faced with stress, plants tend to slowdown or stop growth processes in order to redirect energy toward stressadaptive processes and/or ration available nutrient resources for survival. 58,104,105The visible cessation of growth, as seen under drought and other abiotic stresses 105 is underpinned by a reprogramming of metabolite flux across the entire network of metabolic circuitry. 104While post-translational modifications are key regulatory switches controlling enzyme activity, the downregulation of protein expression serves as a powerful strategy to tone-down or stop flux through selected metabolic pathways.We observed that 43% of the ABA-responsive ECM proteins was down-regulated and the majority were associated with metabolism, cell wall modification and defence/detoxification processes (Table S6).In the TSP, down-regulated proteins constituted 35% of the ABA-responsive proteins and were mainly associated with metabolism and defence/detoxification (Table S5).However, we could not discern any precise theme from the downregulated proteins (Tables S5  and S6).Nevertheless, it is possible the downregulation of protein expression observed in this study might represent aspects of constricting or shutting down certain metabolic pathways.In a way, this may contribute to induction of dormancy, which is necessary until the stress in relieved. 58We thus propose a global systems biology analysis to link these decreases in protein abundance with downstream changes in metabolite content and morphophysiological properties of sorghum plants under ABA treatment with and without water deficit stress.

Genes encoding proteins responsive to ABA in vitro also respond to drought in planta
The above results show the proteomic response of an in vitro sorghum cell culture system to the stress hormone ABA.Our hypothesis is that this provides new insights into what happens in plants responding to drought stress.To test this hypothesis, we used two previously characterized sorghum varieties 52 with distinct drought response phenotypes -SA1441 (drought-tolerant) and ICSB338 (drought-susceptible) and investigated gene expression in drought-stressed root tissue.We selected nine genes encoding ABA-responsive proteins we identified in this study (Tables S5 and S6).This list consisted of up-regulated and downregulated proteins: PR4 (SORBI_3005G169300), PRX3 (SORBI_3002G391300), PRX9 (SORBI_3003G152100), GH19-1 (SORBI_3006G132300), GH19-2 (SORBI_3006G132400), GH9 (SORBI_3003G015700), NLTP (SORBI_3008G030900), LRRP (SORBI_3005G126200) and GELP (SORBI_3010G044500).We found that drought activated the root tissue expression of all genes coding for proteins that were up-regulated by ABA in the cell culture system, except for SORBI_3010G044500, while genes encoding down-regulated proteins were also down-regulated (Figure 4).The magnitude of response for genes encoding upregulated proteins was significantly higher in the drought-tolerant SA1441 sorghum variety than the susceptible ICSB338 variety.In fact, PRX9 and NLTP were not significantly up-regulated in ICSB338 variety, while in SA1441 the up-regulation was statistically significant.Thus, in addition to validating the proteome data, these results also show that the response is at the transcriptional level.More importantly, the results validate the in vitro cell culture system and confirms that ABA-induced responses are recapitulated in plants responding to drought.

Conclusion
The phytohormone ABA 1,3,4 and the plant ECM [40][41][42][43] play key roles during stress-adaptive yet our knowledge of impact of ABA on secreted proteins is minimal.We observed a greater proportion of sorghum-secreted proteins (~32%) that were responsive to ABA than intracellular proteins (~7%), suggesting that the ECM could be an important target of ABA during stress-adaptive responses.The three most dominant groups in the ECM proteome were defense/detoxification (32%), cell wall modification (27%), and proteolysis (15%), suggesting that ABA drives plant defense and redox homeostasis, cell wall remodeling and protein degradation in the ECM.Our results suggest that depending on the prevailing stress condition promoting ABA accumulation, ABAdependent responses could trigger diverse changes in cell metabolism to protect the cells from further stress damage.For example, the plant may stiffen its cell walls to impede total colonization by the invading pathogens or utilize various pathogenesis-related proteins (e.g.peroxidases, chitinases, RNA nucleases, thaumatins, and proteases) for diverse defense activities against the pathogen.Likewise, ABA-regulated cell wall loosening under drought or salinity stress may promote increased cell growth and root elongation to avoid stressful conditions.With such diverse effects of ABA on the secretome, we propose more transgenic studies to validate the roles of these ABA-responsive secreted proteins in susceptible plant species exposed to individual biotic and abiotic stresses and their combinations.
Leucine-rich repeat-containing N-terminal plant-type domain-containing protein a Protein number (N) assigned in ProteinPilot.b Protein accession numbers obtained from the UniProt database searches against sequences of Sorghum bicolor only.c Ratio represents the average fold-change (n = 4) in response to ABA relative to the control.A positive value indicates up-regulation, while a negative value indicates down-regulation.d Standard deviation of the fold-changes (n = 4).e Probability value obtained from a Student's t-test comparing the fold changes between the ABA treatment and the control (n = 4).f Theoretical molecular weight (MW) of each protein as predicted by the Expasy Compute pI/Mw tool on the UniProt database (https://uniprot.org).
repeat-containing N-terminal plant-type domain-containing protein a Protein number (N) assigned in ProteinPilot.b Protein accession numbers obtained from the UniProt database searches against sequences of Sorghum bicolor only.c Ratio represents the average fold-change (n = 4) in response to ABA relative to the control.A positive value indicates up-regulation, while a negative value indicates down-regulation.d Standard deviation of the fold-changes (n = 4).e Probability value obtained from a Student's t-test comparing the fold changes between the ABA treatment and the control (n = 4).f Theoretical molecular weight (MW) of each protein as predicted by the Expasy Compute pI/Mw tool on the UniProt database (https://uniprot.org).

Figure 1 .
Figure 1.Predictions of signal peptides and cellular locations of ABA-responsive proteins of sorghum cell suspension cultures.(a) signal peptide predictions for ABAresponsive secreted proteins were done using the SignalP 6.0 server.(b) gene ontology terms for cellular components of ABA-responsive secreted and total soluble proteins were retrieved from the UniProt database.

Figure 2 .
Figure 2. Biological processes of ABA-responsive secreted and total soluble proteins of sorghum cell suspension cultures.Gene Ontology terms for biological processes were retrieved from the UniProt database.

Figure 3 .
Figure 3. Putative functional groupings of the intracellular and extracellular ABA-responsive proteins of sorghum cell suspension cultures.

Figure 4 .
Figure 4. Gene expression analysis of sorghum root tissue following drought stress.Drought-susceptible ICSB338 and drought-tolerant SA1441 sorghum plants were exposed to drought stress by withholding water for 12 days.Root tissue samples were harvested for gene expression analysis using quantitative reverse transcriptionpolymerase chain reaction.Bars represent mean ± SD (n = 3).*, ** and *** represent statistical significance at p ≤.05, 0.01 and 0,001, respectively using a Student's t-test.

Table 1 .
Summary list of sorghum protein counts obtained after iTRAQ and LC-MS /MS analysis.

Table 2 .
List of ABA-responsive total soluble proteins of white sorghum cell suspension cultures at 1% significance level.

Table 3 .
List of ABA-responsive secreted proteins of white sorghum cell suspension cultures at 1% significance level.